Glycosaminoglycans (GAGs) are large unbranched polysaccharides formed by repeating disaccharide sequences, in which one of the components is always an amino sugar (D-galactosamine or D-glucosamine) and the other component is a uronic acid, such as L-glucuronic or L-iduronic, with the exception of keratan sulfate. Except for hyaluronic acid, all GAGs have sulfate groups, such as O-esters or N-sulfate.
GAGs are part of proteoglycans and lipids. Proteoglycans are a specific type of glycoproteins having at least one GAG chain bound to the protein, and they are classified based on the GAG chain present.
The glycosylation of molecules is a post-translational enzymatic process carried out in the endoplasmic reticulum and Golgi apparatus. Glycosylation reactions are performed by enzymes called glycosyltransferases from monosaccharide precursors of endogenous and exogenous origin.
Diseases relating to congenital alterations in glycosylation include mucopolysaccharidoses (MPS). MPS are a heterogeneous group of innate metabolism errors caused by the deficiency of any of the enzymes necessary for the degradation of GAGs. Non-degraded GAGs are usually partially excreted in the urine, but the rest are accumulated in the lysosomes. This accumulation causes cellular alterations and interferences in other metabolic processes with serious consequences for the human body, leading to cell, tissue, and organ damage.
Seven types of MPS with several subtypes involving 11 specific enzymes have been described up until now. Each type of MPS has nonspecific signs and symptoms, and some alterations considered “characteristic”, although they still require performing a precise diagnosis of these diseases by means of determining the enzyme activity involved, and in the best case scenario, performing molecular identification of the affected gene. They are usually difficult to detect in newborns, and an early and precise diagnosis of MPS is critical in order to provide suitable palliative care, and when possible, a treatment specific for the disease (enzyme replacement therapy, hematopoietic stem cell transplant, among others).
The diagnosis of most MPS is not easy and is mainly based on clinical findings. Once a suspicion is established, the physician may count on certain analytical tests such as urine GAG analysis and quantification of enzyme activity in tissues (blood or fibroblasts). Finally, the physician may request the performance of molecular biology techniques to confirm the possible genetic alteration.
Colorimetric methods measuring the increase in GAG compared to the expected levels of GAGs in normal individuals of the same age have been used for quantifying total GAGs in urine. Several assays in which dimethylmethylene blue (DMB) is used as a dye have been described. GAGs bind to DMB and the compound that is formed is detected by means of spectrophotometry. Nevertheless, isolation of the GAG-DMB complex that is formed does not take place (Whitley C. B. et al. 1989. Clin. Chem., 35(3):374-379; Jong J. G. N. et al. 1992. Clin. Chem., 38(6):803-807). However, they have a high percentage of false positives and false negatives, and due to their low specificity, they do not allow discriminating between the different types of GAG.
Methods for assaying specific GAGs which seek to identify the types of GAGs that are excreted in excess and help to provide a better diagnosis, given that the different types of MPS are associated with the increased excretion of specific GAGs, have also been developed. Those which stand out the most among these methods are chromatographic methods, such as HPLC, which despite being sensitive and specific, are not suitable for massive screening due to the high cost and long analysis time required; ELISA methods which are commercially available for certain types of GAGs, but have not been developed for detecting all GAGs; and tandem mass spectrometry methods that have the drawback of being complex due to the molecular heterogeneity of GAGs and difficulty in applying same for mass screening.
GAG depolymerization methods measuring the hexuronic acid residues that all GAGs have (except for keratan sulfate) have also been developed. This measurement is performed directly in the dialysis liquid, after fractionating the sample in an ECTEOLA cellulose column (separating sulfated GAGs from non-sulfated GAGs), or after precipitating it with cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), or aminoacridine. These methods consume a lot of time and provide barely reproducible results due to the interference of other chromogens, or due to the loss of some GAG fractions in prior treatments.
There are also diseases relating to acquired alterations in glycosylation, such as kidney diseases. Acquired or congenital kidney diseases are a major public health concern worldwide. A recent prospective study conducted in the United States indicates that 54% of adults between 30-49 years of age, 52% of adults between 50-64 years of age, and 42% of adults above 65 years of age, are affected by chronic kidney disease, and an increase between 13.2% and 14.4% is expected in 2020, and an increase of 16.7% is expected in 2030. The diagnosis of said disease is based on measuring a series of clinical parameters in urine (glomerular filtration rate or GFR calculated through the levels of creatinine, cystatin C or inulin, proteinuria, hematuria, etc.), imaging techniques (mainly ultrasounds, computed tomography (CT), or nuclear magnetic resonance (NMR)), and anatomical pathology (by means of biopsies obtained in an invasive manner). All of them have a limited differential diagnostic power and none is capable of anticipating the progression of the kidney disease over time in an accurate and efficient manner. These tools furthermore have limited specificity and sensitivity.
There is therefore a need for new methods for detecting the fractions bound to or associated with GAGs which overcome the drawbacks of the methods known up until now, which allow a quick, sensitive, reliable, cost-effective and less invasive diagnosis in early stages of the disease, which can be used for massive screening in newborns, and which allow detecting and/or prognosticating diseases such as MPS or a kidney disease.